ALBERT

Description: Mixed Lineage Leukemia gene rearrangements (MLL-r) account for nearly 10% of human acute leukemia cases and are generally associated with poor prognosis. Previous studies have revealed an essential role of the histone H3K79 methyltransferase Disruptor of Telomeric Silencing-1 Like (DOT1L) in MLL-r leukemogenesis. Our recent report (Chen et al. 2015 Nature Medicine) further identified a role for histone acetylation in DOT1L dependent gene expression driven by MLL-fusion proteins including MEIS1 and HOXA cluster genes. A first-in-human Phase I clinical trial demonstrated clinical activity of DOT1L inhibition in MLL-r leukemia patients, thus providing a potential opportunity for treating these malignant diseases. Nevertheless, the incomplete silencing of the leukemic program by only targeting DOT1L motivates the need for additional and perhaps combinational approaches to improve therapies against MLL-r leukemias. To enhance the efficacy of DOT1L inhibition, we sought to identify genes whose suppression would synergize with the DOT1L inhibitors to suppress the proliferation of mouse bone marrow progenitors transformed with MLL-AF9. We conducted a pooled RNAi screen using a customized library composed of 2,252 shRNA targeting 468 epigenetic regulators (i.e. writers, readers, and erasers of chromatin modifications; Fig 1). The integrated shRNA sequences were assessed using high-throughput sequencing. By comparing the change in frequency of each shRNA construct cultured in control vs. an IC50 DOT1L inhibitor EPZ4777, we identified several candidate modulators of DOT1L dependency, which had multiple shRNAs selectivity depleted only in the DOT1L suppressed condition. Notably, using a network correlation study, we found that one of the top candidate genes Plant Homeodomain Finger Protein 20 (PHF20) is highly associated with histone acetylation in the mammalian epigenome. Knockdown of PHF20 drastically increased the sensitivity of MLL-AF9 leukemic blasts to DOT1L inhibitors through enhanced myeloid differentiation and reduced cell proliferation, colony formation, and re-plating capacity. Similar phenotypes were also observed in PHF20-deficient MLL-AF9 cells generated by CRISPR/Cas9-mediated gene knockout. PHF20 is an epigenetic adaptor protein that has no predicted enzymatic activity. To investigate the role of PHF20, we conducted a CRISPR functional domain screen and identified the requirement of the chromatin reader domains in PHF20, including the Tudor domains and the PHD-finger, in supporting the survival of MLL-r leukemic cells upon DOT1L inhibition. We also performed RNA-seq and found that suppression of PHF20 facilitated the silencing of the MLL-AF9 leukemic program induced by DOT1L inhibitor treatment. Chromatin immunoprecipitation and sequencing (ChIP-seq) analyses validated that PHF20 contributes to the maintenance of histone acetylation including H3K9ac and H4K16ac at MLL-AF9 target loci. In line with the profound loss of histone acetylation at MLL-AF9 target loci in PHF20-depleted cells, we found that knockdown of a known PHF20 interacting partner KAT8 (a histone acetyltransferase; also known as MOF or MYST1) phenocopies the effects observed in PHF20-knockdown cells. Finally, we showed that pharmacological inhibition of DOT1L and KAT8 synergistically suppresses the proliferation and survival of MLL-AF9 leukemic cells. These data collectively highlight the involvement of a novel DOT1L-PHF20-KAT8 axis in mammalian gene regulation and MLL-r leukemogenesis. In summary, our studies show that MLL-rearrangements may drive leukemic transformation by coordinating an epigenetic network involving several histone modifications associated with gene transcription (e.g. H3K79 methylation and H3K9/H4K16 acetylation). Our results also suggest that simultaneous targeting of multiple components of this epigenetic feed-forward loop including DOT1L and PHF20/KAT8 may provide a novel and more effective approach against MLL-r leukemia. Disclosures Bradner: Novartis Institutes for BioMedical Research: Employment. Armstrong:Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.

Description: Background: Activating mutations of NRAS and KRAS genes are common in newly diagnosed acute myeloid leukemia (AML), occurring in 11-16% and 4-5% of patients, respectively. RAS mutations are frequently acquired at time of progression from MDS to AML and are associated with poor survival. Next generation sequencing (NGS) at diagnosis and during complete remission has shown that RAS mutations have high clearance rates with induction chemotherapy. In the CALGB 8525 study, RAS-mutant younger patients (age

Description: Background Measurable residual disease (MRD) is associated with inferior outcomes in patients with acute myeloid leukemia (AML). MRD monitoring enhances risk stratification and may guide therapeutic intervention. Post-induction MRD is frequently cleared with further therapy and the clearance may lead to better outcomes. In contrast, persistent MRD is associated with poor outcomes. At present it is not possible to predict which patients are likely to clear MRD with further therapy. Here we report a simple, objective, widely applicable and quantitative MFC approach using the ratio of blast/PDC to predict persistent MRD and poor outcomes in AML. Patients and Methods A cohort of 136 adult patients with a confirmed diagnosis of AML by WHO criteria who underwent standard induction therapy at a single center between 4/2014 and 9/2017 was initially included. 69 patients achieved complete morphologic remission (36 MRD-neg. and 33 MRD-pos.). MRD status was assessed by MFC using a different from normal (DfN) approach. PDC were quantified as the percent of total WBC by flow cytometry based on low side scatter, moderate CD45, CD303, bright CD123 and HLA-DR expression. Results The proportion of PDC was markedly decreased in patients with AML (≥20% blasts) (N=136) with a median of 0.016% (interquartile range IQR: 0.0019%-0.071%, Figure 1A), more than 10-fold lower than observed in normal controls (median 0.23%, IQR 0.17%-0.34%) (N=20). While there was no difference between MRD-neg. and normal control groups (median 0.31%, IQR: 0.17%-0.49%; vs. 0.28%, IQR: 0.17%-0.34%), MRD-pos. group had significantly reduced PDC proportion compared to the control (median 0.074%, IQR: 0.022%-0.33%, Wilcoxon rank sum, p=0.019). In an attempt to achieve better separation and to eliminate possible effects of hemodilution, the ratio of blast/PDC was calculated by using the proportions of blasts and PDCs out of total WBCs as quantitated by flow cytometry. A cut-off threshold of the blast/PDC ratio of 10 was chosen to separate each group (Figure 1B). Importantly, a ratio cut-off of 10 had a corresponding specificity of 97.4% for predicting MRD positivity status. MRD positivity was significantly associated with inferior overall survival (OS) and relapse-free survival (RFS) in our study cohort (OS HR 4.11 (95% CI: 1.30-13.03), p=0.016; RFS HR 4.20 (95% CI: 1.49-11.82), p=0.007, Figure 1C and D). The 2-year cumulative incidence of relapse in the MRD-neg. group compared to MRD-pos. group was 10% (95% CI: 2-24%) vs. 37% (95% CI: 18-56%, p=0.014). Importantly, blast/PDC ratio ≥10 was also strongly associated with inferior OS and RFS (OS HR 3.12 (95% CI: 1.13-8.60), p= 0.028; RFS HR 4.05 (95% CI: 1.63-10.11), p=0.003, Figure 1E and F), which is similar in magnitude to MRD positivity. Furthermore, MRD-pos. patients with blast/PDC ratio

Description: Background: Chromosomal rearrangements involving the 11q23 locus, resulting in fusions of the Mixed Lineage Leukemia (MLL) gene, are found in 5-10% of adult patients with acute myeloid leukemia and can represent a poor prognostic feature. Patients with this subtype of AML often respond well to standard induction chemotherapy but frequently relapse, even after allogeneic hematopoietic stem cell transplantation. Effective therapy options in the relapsed / refractory setting for this high-risk group represent an urgent unmet clinical need. In MLL-rearranged AML, recruitment of disruptor of telomeric silencing 1-like (DOT1L), a histone H3 lysine 79 (H3K79) methyltransferase, and subsequent changes in methylation of downstream targets HOXA9 and Meis1 is central to leukemogenesis. DOT1L-mediated expression of MLL target genes is critical to developing leukemia in a murine model and inhibition of DOT1L suppressed downstream expression of MLL target genes. Pinometostat is a potent and selective small molecule inhibitor of DOT1L methyltransferase activity. Use of pinometostat in a continuous IV infusion in a rat xenograft model of MLL-rearranged leukemia showed complete, sustained tumor regressions without significant appreciable toxicities. A recent single-agent, phase I trial evaluated pinometostat in R/R patients with MLL-R myeloid malignancies. This study included 43 patients with MLL-R AML and observed that the drug was generally well-tolerated. Responses to pinometostat included one patient with morphologic CR, one with cytogenetic CR, three with resolution of leukemia cutis, and nine with signs of differentiation / leukocytosis. Promoter hypermethylation contributes to the dysregulation of MLL-R target genes HOXA9 and Meis1, and this effect was reversed upon treatment with the hypomethylating agent azacitidine. As such, this combination may lead to a more robust response in this patient population. We now seek to combine the novel, targeted agent pinometostat with azacitidine in R/R MLL-R AML. Study design & methods: We are conducting an open-label, single-arm, phase Ib / II study that will enroll 36-48 patients with R/R MLL-R AML to evaluate the tolerability and preliminary efficacy of pinometostat in combination with azacitidine. Since MLL-R AML is a rare disease and is seen in only 5-10% of patients with AML, we are looking to collaborate with other centers through the National Cancer Institute Experimental Therapeutics Clinical Trials Network (ETCTN) to meet our accrual target. The study is active nationally and enrolling patients (NCT03701295). The presence of the MLL-R will be confirmed locally by FISH or cytogenetics. Adult patients who are refractory to two courses of induction, relapse after CR, or elect not to pursue induction therapy are considered eligible. Patients who have previously undergone HSCT or who have well-controlled HIV are also candidates. Pinometostat will be given by continuous IV infusion via portable pump. Azacitidine will be administered at 75mg/m2 daily over 7 days at the start of each 28-day cycle. Patients will be followed on trial for a total of 6 cycles, with bone marrow biopsies done for assessment after cycles 1, 3, and 6. The dose of pinometostat will be escalated following a standard 3+3 design, and the primary endpoint for the phase Ib portion will be safety and tolerability. The primary endpoint for the phase II will be response to combination therapy as defined by the 2017 European Leukemia Network guidelines. Accrual will proceed as per a Simon two-stage minimax design. Integrative correlative analyses will include genomics, changes in DOT1L-mediated methylation by H3K79 ELISA, and qPCR of HOXA9 and Meis1. PK studies and azacitidine incorporation and DNA methylation studies will also be performed. Disclosures Cai: Imago Biosciences, Inc.: Consultancy. Armstrong:AstraZeneca: Research Funding; Epizyme, Inc.: Consultancy, Equity Ownership; Imago Biosciences, Inc.: Consultancy, Equity Ownership; Cyteir Therapeutics: Consultancy, Equity Ownership; C4 Therapeutics: Consultancy, Equity Ownership; Syros Pharmaceuticals: Consultancy, Equity Ownership; OxStem Oncology: Consultancy, Equity Ownership; Accent Therapeutics: Consultancy, Equity Ownership; Mana Therapeutics: Consultancy, Equity Ownership; Novartis: Research Funding; Janssen: Research Funding. Rudek:RenovoRX: Research Funding; Taiho: Research Funding; Celgene: Research Funding; Cullinan Apollo: Research Funding. Tallman:Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; UpToDate: Patents & Royalties; ADC Therapeutics: Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; Cellerant: Research Funding; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; ADC Therapeutics: Research Funding; ADC Therapeutics: Research Funding; ADC Therapeutics: Research Funding; ADC Therapeutics: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Biosight: Research Funding; Biosight: Research Funding; Biosight: Research Funding; Cellerant: Research Funding; Cellerant: Research Funding; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stein:PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees.

Description: Cytotoxic lymphocytes, which include CD4+ and CD8+ T cells as well as NK cells, use the granule exocytosis pathway to kill virus-infected and tumor cells. Previous studies have characterized the expression of many genes in this pathway (e.g. perforin, granzyme A, granzyme B, etc.), although no reagent has been developed to measure granzyme C protein at the single-cell level. The murine granzyme C gene, orthologous to human granzyme H, lies 24.2Kb directly downstream from granzyme B in the granzyme B gene cluster. Recombinant granzyme C rapidly induces target cell death in a manner distinct from granzyme A- or B-induced death. To further understand the regulation and function of murine granzyme C, we developed a granzyme C-specific monoclonal antibody and used flow cytometry to examine the expression of granzyme C in resting and activated murine cytotoxic lymphocyte compartments. Naive CD4+ and CD8+ T cells express little or no granzyme C (0.1 + 0.1% of cells are positive). After activation of splenocytes with plate-bound CD3 and CD28 agonistic antibodies for 4 days, a small percentage of CD4+ (2.3 + 1.0% positive) and CD8+ (6.6 + 0.3% positive) T cells express granzyme C. However, only 24 hours later, almost all CD4+ (96.7 + 2.7%) and CD8+ (98.7 + 1.4%) T cells express granzyme C. Granzyme B co-staining revealed that granzyme B is detectable in both CD4+ and CD8+ T cells at least 48 hours before granzyme C is expressed. Furthermore, we employed a fully mismatched GVHD mouse model in order to examine T cell expression of granzymes B and C in vivo; similarly, granzyme B expression preceded granzyme C by at least 48 hours. In addition, CD4+Foxp3+ regulatory T cells also expressed granzyme C in this model. Murine NK cell granzyme C mRNA expression was assessed using Affymetrix microarrays (MOE430v2) at rest and following IL-15 activation. Resting NK cells express minimal granzyme C mRNA (mean gzmC probe set signal intensity + SD [n=3]): 356 + 202, which increased after IL-15 activation as follows: 4,180 + 1,106 (day 1), 60,788 + 8,455 (day 2), 167,448 + 26,398 (day 4), and 178,451+14,037 (day 6). Utilizing our granzyme C-specific mAb, we showed that very few resting murine NK cells express granzyme C protein (2.3 + 0.4% positive). Consistent with the mRNA data, the percentage of granzyme C-expressing NK cells was substantially increased after 3 days of IL-15 activation (88.8 + 5.2% positive). In contrast, granzyme B mRNA levels are high in resting NK cells (41,208 + 2,534), and increase only incrementally with IL-15 treatment (Fehniger et al., Immunity 2007 Jun;26(6):798–811). Granzyme B protein expression is controlled at a post-transcriptional level in NK cells, whereas granzyme C expression is transcriptionally regulated. Taken together, our findings show that the mouse granzyme B and C genes, despite being only 24.2Kb apart, are activated in cytotoxic lymphocytes with different kinetics; moreover, granzyme B and C protein abundance is controlled by completely different mechanisms in NK cells. These data suggest that the granzyme genes are differentially regulated in lymphocyte compartments, and that this regulation may be relevant for how cytotoxic lymphocytes function.

Description: Key Points LSD1 inhibition induces a global increase in chromatin accessibility, whereas DOT1L inhibition induces global decreases in accessibility. Perturbation of PU.1 and C/EBPα expression renders AML cells more resistant to LSD1 inhibition.

Description: Prior studies have shown that the cell of origin of acute myeloid leukemia (AML) initiation is an important determinant of therapeutic sensitivity. MLL-rearranged leukemias in which the fusion is acquired in hematopoietic stem cells (HSC) are less sensitive to chemotherapy and express high levels of the oncogenic transcription factor, Evi1, when compared to leukemias which are initiated in granulocyte-macrophage progenitors (GMP). However, the mechanisms governing how cell-of-origin modulates therapeutic response have not been delineated. Here, we describe a functional link that ties therapeutic sensitivity of MLL-AF9 leukemias to both chemotherapy and pharmacologic inhibitors of lysine-specific demethylase 1 (LSD1) - currently under investigation in clinical trials - to AML cell of origin via a novel mechanism that modulates p53 protein stability and activity through Evi1. This Evi1-dependent mechanism revealed a therapeutic vulnerability in the resistant HSC-derived leukemias that could be targeted with the BCL2 inhibitor venetoclax, which overcame resistance when combined with LSD1 inhibition. Murine HSC-derived MLL-AF9 leukemias exhibited markedly reduced sensitivity to the LSD1 inhibitor, IMG-7289, when compared to GMP-derived MLL-AF9 leukemias, in vitro and in vivo. Consistent with previously published reports, HSC-derived leukemias exhibited several hundred-fold higher expression of EVI1 mRNA relative to GMP-derived leukemias in both mouse and human MLL-rearranged leukemia models; the differential EVI1 expression mirrors expression patterns of this key hematopoietic regulator in normal HSCs and GMPs. H3K27me3 chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) revealed that the EVI1 locus is silenced by Polycomb repression in GMP-derived leukemias, and this repression was also observed in EVI1low primary AML patient samples. Knockdown of Evi1 via shRNA sensitized HSC-derived leukemias to LSD1 inhibition. In vitro assays revealed induction of apoptosis in GMP-derived leukemias - but not in HSC-derived leukemias - after treatment with IMG-7289. HSC-derived leukemias also exhibited decreased apoptotic priming assessed through functional BH3 profiling assays as well as blunted p53 transcriptional output. These data suggested that cell-of-origin led to differential p53 activity and therapeutic response in AMLs driven by the same fusion gene initiated from different stem/progenitor populations. The diminished p53 activity in HSC-derived leukemias was associated with reduced p53 protein abundance both at steady-state and after LSD1 inhibitor treatment compared to GMP-derived leukemias. Quantification of p53 mRNA did not show differential p53 expression, including after LSD1 inhibition, in HSC vs. GMP-derived leukemias implicating post-transcriptional regulatory mechanisms that underlie this cell-of-origin mediated phenotype. We found that modulation of Evi1 expression resulted in altered p53 protein stability: specifically, (1) shRNA-mediated knockdown of Evi1 in HSC-derived leukemias increased p53 protein stability and (2) overexpression of Evi1 blunted doxorubicin-induced p53 protein stability. Moreover, p53 loss-of-function in Evi1low GMP-derived MLL-AF9 leukemias induced resistance to LSD1 inhibition. By contrast, Evi1high HSC-derived leukemias exposed to the BCL2 inhibitor venetoclax in vivo were sensitized to LSD1 inhibition, resulting in enhanced apoptosis and greater reductions in disease burden, observations that we observed in patients with Evi1high AML treated with venetoclax. Our findings describe how the cell of origin of p53 wild-type cancers can differentially modulate p53 function and therapeutic response and provide a mechanistic rationale for therapies aimed to circumvent this resistance mechanism. Disclosures Cai: Imago Biosciences, Inc.: Consultancy. Goldberg:Celgene: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Pfizer: Research Funding; Arog Pharmaceuticals: Research Funding; ADC Therapeutics: Research Funding; American Society of Clinical Oncology: Research Funding; Abbvie: Consultancy; Abbvie: Research Funding; American Society of Hematology: Research Funding; DAVA Oncology: Honoraria. Stein:Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Tallman:Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; ADC Therapeutics: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; UpToDate: Patents & Royalties; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Cellerant: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; UpToDate: Patents & Royalties; Cellerant: Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellerant: Research Funding; Biosight: Research Funding; Biosight: Research Funding; ADC Therapeutics: Research Funding; ADC Therapeutics: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Cellerant: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellerant: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biosight: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Biosight: Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lowe:ORIC pharmaceuticals: Consultancy, Equity Ownership; Mirimus: Consultancy, Equity Ownership; Constellation Pharma: Consultancy, Equity Ownership; Petra Pharmaceuticals: Consultancy, Equity Ownership; PMV Pharmaceuticals: Consultancy, Equity Ownership; Faeth Therapeutics: Consultancy, Equity Ownership; Blueprint Medicines: Consultancy, Equity Ownership. Rienhoff:Imago Biosciences: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Levine:Loxo: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Amgen: Honoraria; Celgene: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armstrong:AstraZeneca: Research Funding; Epizyme, Inc.: Consultancy, Equity Ownership; Imago Biosciences, Inc.: Consultancy, Equity Ownership; Cyteir Therapeutics: Consultancy, Equity Ownership; C4 Therapeutics: Consultancy, Equity Ownership; Syros Pharmaceuticals: Consultancy, Equity Ownership; OxStem Oncology: Consultancy, Equity Ownership; Accent Therapeutics: Consultancy, Equity Ownership; Mana Therapeutics: Consultancy, Equity Ownership; Novartis: Research Funding; Janssen: Research Funding.

Description: Regulatory T (Treg) cells can suppress a wide variety of immune responses, including antitumor and alloimmune responses. The mechanisms by which Treg cells mediate their suppressive effects depend on the context of their activation. We previously reported that granzyme B is important for Treg cell–mediated suppression of antitumor immune responses. We therefore hypothesized that granzyme B may likewise be important for suppression of graft-versus-host disease (GVHD). We found that allogeneic mismatch induces the expression of granzyme B in mixed lymphocyte reactions and in a model of graft-versus-host disease (GVHD). However, wild-type and granzyme B–deficient Treg cells were equally able to suppress effector T (Teff) cell proliferation driven by multiple stimuli, including allogeneicantigen-presenting cells. Surprisingly, adoptive transfer of granzyme B–deficient Treg cells prevented GVHD lethality, suppressed serum cytokine production in vivo, and prevented target organ damage. These data contrast strikingly with our previous study, which demonstrated that granzyme B plays a nonredundant role in Treg cell–mediated suppression of antitumor responses. Taken together, these findings suggest that targeting specific Treg cell–suppressive mechanisms, such as granzyme B, may be therapeutically beneficial for segregating GVHD and graft-versus-tumor immune responses.

Description: Background: MRD is a powerful prognostic factor in AML. Emerging data indicate that allogeneic stem cell transplant (alloSCT) with MRD results in outcomes equivalently poor to alloSCT with morphologic AML (Araki et al., JCO 2016). Genomic predictors of MRD are unclear, and relative efficacy of therapies for MRD remains elusive. Objectives: Here we provide an integrated analysis of responses for 163 patients (pts) who underwent induction chemotherapy with baseline next-generation sequencing (NGS) followed by serial immunophenotypic monitoring for MRD. Methods:163 patients starting in April 2014 who underwent induction chemotherapy at Memorial Sloan Kettering Cancer Center were retrospectively studied. All received anthracycline + cytarabine, with or without investigational agents. Immunophenotypic MRD was identified in bone marrow aspirates (BMA) by multiparameter flow cytometry. Any level of residual disease was considered MRD+. Molecular analysis was obtained from pre-induction BMA by NGS using 28 or 49 gene panels. Cytogenetics/FISH were performed using standard techniques. Results: Patient characteristics are in Table 1. 7/163 (4.9%) died within 30 days of induction.153 pts had BM biopsy after induction prior to further therapy. 124/153 underwent flow after induction. 65/124 (52.4%) achieved CR/CRi after induction alone, 31/124 (25%) MRD+CR/CRi, and 34/124 (27.4%) MRD-CR/CRi. Pre-induction molecular analysis from 126 suggests that certain cytogenetic and molecular abnormalities correlate with achievement of MRD-CR. (Figure 1) Only 2/25 (8%) with RUNX1, 0/13 with SF3B1, and 0/11 with TP53 mutations achieved MRD-CR/CRi as best response after 1 cycle of induction. Only 3 additional RUNX1, 2 SF3B1, and 0/11 TP53 achieved MRD-CR/CRi as best response after a second cycle of therapy. In contrast, 7/8 with CBF AML (inv16 and no KIT mutation, n=4) or (t(8;21), n=3) achieved MRD-CR/CRi (n=5) or CR without flow (n=2) after 1 cycle of induction. 91/163 (55.8%) underwent alloSCT following induction or additional therapy. Post-alloSCT follow-up indicates potential value in converting MRD+ to MRD-. 84/91 were evaluable for MRD with flow cytometry prior to alloSCT. 41/84 (48.8%) were MRD-, 30/84 (35.7%) MRD+, and 13/84 (15.4%) persistent AML. 13/41 (31.7%) MRD-pre-alloSCT were MRD- post-induction. 28/41 (68.2%) MRD+ or persistent AML converted to MRD- prior to alloSCT following additional therapy. 23/29 MRD+CR/CRi pts after induction were intermediate/unfavorable and therefore transplant candidates. 19/23 MRD+CR/CRi intermediate/unfavorable underwent transplant (9 without post-induction therapy, 10 after consolidation), while 4 did not proceed to transplant due to relapse after induction (n=1), relapse after consolidation (n=2), and patient preference. There was no significant difference in post-transplant OS between early MRD-CR immediately following induction and later conversion to MRD-CR prior to alloSCT (Figure 1B). Post-transplant analysis reveals that most pts who enter transplant with persistent AML (n=13) or MRD+ (n=30) clear MRD (30/43, 69.7%) by the first post-transplant BM (median 32 days, Figure 1C). Despite initial post-transplant MRD clearance, pts who entered alloSCT with persistent AML or MRD+ had poorer post-transplant OS compared to pts who entered alloSCT with MRD- (p=0.02, Figure 1D). Conclusion: Our data show that AML pts with specific molecular mutations (RUNX1, SF3B1, and TP53) are unlikely to achieve MRD-CR/CRi after induction chemotherapy. We further show that additional therapy such as consolidation may be advantageous for some MRD+ pts to achieve MRD-CR prior to alloSCT, although others remain resistant to MRD clearance. Post-transplant OS is improved in pts who are MRD- at time of transplant, regardless of whether they required additional therapy beyond induction to achieve this state. Our results suggest that development of MRD-eradicating therapies after AML induction has the potential to improve post-transplant outcomes. Disclosures Goldberg: AROG: Research Funding; Pfizer: Research Funding; Celgene: Consultancy. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Perales:Takeda: Other: Personal fees; Merck: Other: Personal fees; Abbvie: Other: Personal fees; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support; Novartis: Other: Personal fees. Tallman:ADC Therapeutics: Research Funding; Daiichi-Sankyo: Other: Advisory board; Orsenix: Other: Advisory board; Cellerant: Research Funding; BioSight: Other: Advisory board; AROG: Research Funding; AbbVie: Research Funding.